Use of metal-based contrast agents for in vivo MR and CT imaging of phagocytic cells in neurological pathologies.

The activation of phagocytic cells is a hallmark of many neurological diseases. Imaging them in their 3-dimensional cerebral environment over time is crucial to better understand their role in disease pathogenesis and to monitor their potential therapeutic effects. Phagocytic cells have the ability to internalize metal-based contrast agents both in vitro and in vivo and can thus be tracked by magnetic resonance imaging (MRI) or computed tomography (CT). In this review article, we summarize the different labelling strategies, contrast agents, and in vivo imaging modalities that can be used to monitor cells with phagocytic activity in the central nervous system using MRI and CT, with a focus on clinical applications. Metal-based nanoparticle contrast agents such as gadolinium, gold and iron are ideal candidates for these applications as they have favourable magnetic and/or radiopaque properties and can be fine-tuned for optimal uptake by phagocytic cells. However, they also come with downsides due to their potential toxicity, especially in the brain where they might accumulate. We therefore conclude our review by discussing the pitfalls, safety and potential for clinical translation of these metal-based neuroimaging techniques. Early results in patients with neuropathologies such as multiple sclerosis, stroke, trauma, cerebral aneurysm and glioblastoma are promising. If the challenges represented by safety issues are overcome, phagocytic cells imaging will be a very valuable tool for studying and understanding the inflammatory response and evaluating treatments that aim at mitigating this response in patients with neurological diseases.

Brain virtual histology with X-ray phase-contrast tomography Part I: whole-brain myelin mapping in white-matter injury models.

White-matter injury leads to severe functional loss in many neurological diseases. Myelin staining on histological samples is the most common technique to investigate white-matter fibers. However, tissue processing and sectioning may affect the reliability of 3D volumetric assessments. The purpose of this study was to propose an approach that enables myelin fibers to be mapped in the whole rodent brain with microscopic resolution and without the need for strenuous staining. With this aim, we coupled in-line (propagation-based) X-ray phase-contrast tomography (XPCT) to ethanol-induced brain sample dehydration. We here provide the proof-of-concept that this approach enhances myelinated axons in rodent and human brain tissue. In addition, we demonstrated that white-matter injuries could be detected and quantified with this approach, using three animal models: ischemic stroke, premature birth and multiple sclerosis. Furthermore, in analogy to diffusion tensor imaging (DTI), we retrieved fiber directions and DTI-like diffusion metrics from our XPCT data to quantitatively characterize white-matter microstructure. Finally, we showed that this non-destructive approach was compatible with subsequent complementary brain sample analysis by conventional histology. In-line XPCT might thus become a novel gold-standard for investigating white-matter injury in the intact brain. This is Part I of a series of two articles reporting the value of in-line XPCT for virtual histology of the brain; Part II shows how in-line XPCT enables the whole-brain 3D morphometric analysis of amyloid- ß (A ß ) plaques.

Neurofunctional and neuroimaging readouts for designing a preclinical stem-cell therapy trial in experimental stroke.

With the aim of designing a preclinical study evaluating an intracerebral cell-based therapy for stroke, an observational study was performed in the rat suture model of ischemic stroke. Objectives were threefold: (i) to characterize neurofunctional and imaging readouts in the first weeks following transient ischemic stroke, according to lesion subtype (hypothalamic, striatal, corticostriatal); (ii) to confirm that intracerebral administration does not negatively impact these readouts; and (iii) to calculate sample sizes for a future therapeutic trial using these readouts as endpoints. Our results suggested that the most relevant endpoints were side bias (staircase test) and axial diffusivity (AD) (diffusion tensor imaging). Hypothalamic-only lesions did not affect those parameters, which were close to normal. Side bias in striatal lesions reached near-normal levels within 2 weeks, while rats with corticostriatal lesions remained impaired until week 14. AD values were decreased at 4 days and increased at 5 weeks post-surgery, with a subtype gradient: hypothalamic < striatal < corticostriatal. Intracerebral administration did not impact these readouts. After sample size calculation (18-147 rats per group according to the endpoint considered), we conclude that a therapeutic trial based on both readouts would be feasible only in the framework of a multicenter trial.

Multimodal Imaging with NanoGd Reveals Spatiotemporal Features of Neuroinflammation after Experimental Stroke.

The purpose of this study is to propose and validate a preclinical in vivo magnetic resonance imaging (MRI) tool to monitor neuroinflammation following ischemic stroke, based on injection of a novel multimodal nanoprobe, NanoGd, specifically designed for internalization by phagocytic cells. First, it is verified that NanoGd is efficiently internalized by microglia in vitro. In vivo MRI coupled with intravenous injection of NanoGd in a permanent middle cerebral artery occlusion mouse model results in hypointense signals in the ischemic lesion. In these mice, longitudinal two-photon intravital microscopy shows NanoGd internalization by activated CX3CR1-GFP/+ cells. Ex vivo analysis, including phase contrast imaging with synchrotron X-ray, histochemistry, and transmission electron microscopy corroborate NanoGd accumulation within the ischemic lesion and uptake by immune phagocytic cells. Taken together, these results confirm the potential of NanoGd-enhanced MRI as an imaging biomarker of neuroinflammation at the subacute stage of ischemic stroke. As far as it is known, this work is the first to decipher the working mechanism of MR signals induced by a nanoparticle passively targeted at phagocytic cells by performing intravital microscopy back-to-back with MRI. Furthermore, using a gadolinium-based rather than an iron-based contrast agent raises future perspectives for the development of molecular imaging with emerging computed tomography technologies.

Hybrid multimodal contrast agent for multiscale in vivo investigation of neuroinflammation.

Neuroinflammation is a process common to several brain pathologies. Despites its medical relevance, it still remains poorly understood; there is therefore a need to develop new in vivo preclinical imaging strategies to monitor inflammatory processes longitudinally. We here present the development of a hybrid imaging nanoprobe named NP3, that was specifically designed to get internalized by phagocytic cells and imaged in vivo with MRI and bi-photon microscopy. NP3 is composed of a 16 nm core of gadolinium fluoride (GdF3), coated with bisphosphonate polyethylene glycol (PEG) and functionalized with a Lemke-type fluorophore. It has a hydrodynamic diameter of 28 ± 8 nm and a zeta potential of -42 ± 6 mV. The MR relaxivity ratio at 7 T is r1/r2 = 20; therefore, NP3 is well suited as a T2/T2* contrast agent. In vitro cytotoxicity assessments performed on four human cell lines revealed no toxic effects of NP3. In addition, NP3 is internalized by macrophages in vitro without inducing inflammation or cytotoxicity. In vivo, uptake of NP3 has been observed in the spleen and the liver. NP3 has a prolonged vascular remanence, which is an advantage for macrophage uptake in vivo. The proof-of-concept that NP3 may be used as a contrast agent targeting phagocytic cells is provided in an animal model of ischemic stroke in transgenic CX3CR1-GFP/+ mice using three complementary imaging modalities: MRI, intravital two-photon microscopy and phase contrast imaging with synchrotron X-rays. In summary, NP3 is a promising preclinical tool for the multiscale and multimodal investigation of neuroinflammation.

Multicolor spectral photon counting CT monitors and quantifies therapeutic cells and their encapsulating scaffold in a model of brain damage.

Rationale & aim: Various types of cell therapies are currently under investigation for the treatment of ischemic stroke patients. To bridge the gap between cell administration and therapeutic outcome, there is a need for non-invasive monitoring of these innovative therapeutic approaches. Spectral photon counting computed tomography (SPCCT) is a new imaging modality that may be suitable for cell tracking. SPCCT is the next generation of clinical CT that allows the selective visualization and quantification of multiple contrast agents. The aims of this study are: (i) to demonstrate the feasibility of using SPCCT to longitudinally monitor and quantify therapeutic cells, i.e. bone marrow-derived M2-polarized macrophages transplanted in rats with brain damage; and (ii) to evaluate the potential of this approach to discriminate M2-polarized macrophages from their encapsulating scaffold. Methods: Twenty one rats received an intralesional transplantation of bone marrow-derived M2-polarized macrophages. In the first set of experiments, cells were labeled with gold nanoparticles and tracked for up to two weeks post-injection in a monocolor study via gold K-edge imaging. In the second set of experiments, the same protocol was repeated for a bicolor study, in which the labeled cells are embedded in iodine nanoparticle-labeled scaffold. The amount of gold in the brain was longitudinally quantified using gold K-edge images reconstructed from SPCCT acquisition. Animals were sacrificed at different time points post-injection, and ICP-OES was used to validate the accuracy of gold quantification from SPCCT imaging. Results: The feasibility of therapeutic cell tracking was successfully demonstrated in brain-damaged rats with SPCCT imaging. The imaging modality enabled cell monitoring for up to 2 weeks post-injection, in a specific and quantitative manner. Differentiation of labeled cells and their embedding scaffold was also feasible with SPCCT imaging, with a detection limit as low as 5,000 cells in a voxel of 250 × 250 × 250 µm in dimension in vivo. Conclusion: Multicolor SPCCT is an innovative translational imaging tool that allows monitoring and quantification of therapeutic cells and their encapsulating scaffold transplanted in the damaged rat brain.

MRI coupled with clinically-applicable iron oxide nanoparticles reveals choroid plexus involvement in a murine model of neuroinflammation.

Choroid plexus (ChPs) are involved in the early inflammatory response that occurs in many brain disorders. However, the activation of immune cells within the ChPs in response to neuroinflammation is still largely unexplored in-vivo. There is therefore a crucial need for developing imaging tool that would allow the non-invasive monitoring of ChP involvement in these diseases. Magnetic resonance imaging (MRI) coupled with superparamagnetic particles of iron oxide (SPIO) is a minimally invasive technique allowing to track phagocytic cells in inflammatory diseases. Our aim was to investigate the potential of ultrasmall SPIO (USPIO)-enhanced MRI to monitor ChP involvement in-vivo in a mouse model of neuroinflammation obtained by intraperitoneal administration of lipopolysaccharide. Using high resolution MRI, we identified marked USPIO-related signal drops in the ChPs of animals with neuroinflammation compared to controls. We confirmed these results quantitatively using a 4-points grading system. Ex-vivo analysis confirmed USPIO accumulation within the ChP stroma and their uptake by immune cells. We validated the translational potential of our approach using the clinically-applicable USPIO Ferumoxytol. MR imaging of USPIO accumulation within the ChPs may serve as an imaging biomarker to study ChP involvement in neuroinflammatory disorders that could be applied in a straightforward way in clinical practice.

Clinical Imaging of Choroid Plexus in Health and in Brain Disorders: A Mini-Review.

The choroid plexuses (ChPs) perform indispensable functions for the development, maintenance and functioning of the brain. Although they have gained considerable interest in the last years, their involvement in brain disorders is still largely unknown, notably because their deep location inside the brain hampers non-invasive investigations. Imaging tools have become instrumental to the diagnosis and pathophysiological study of neurological and neuropsychiatric diseases. This review summarizes the knowledge that has been gathered from the clinical imaging of ChPs in health and brain disorders not related to ChP pathologies. Results are discussed in the light of pre-clinical imaging studies. As seen in this review, to date, most clinical imaging studies of ChPs have used disease-free human subjects to demonstrate the value of different imaging biomarkers (ChP size, perfusion/permeability, glucose metabolism, inflammation), sometimes combined with the study of normal aging. Although very few studies have actually tested the value of ChP imaging biomarkers in patients with brain disorders, these pioneer studies identified ChP changes that are promising data for a better understanding and follow-up of diseases such as schizophrenia, epilepsy and Alzheimer's disease. Imaging of immune cell trafficking at the ChPs has remained limited to pre-clinical studies so far but has the potential to be translated in patients for example using MRI coupled with the injection of iron oxide nanoparticles. Future investigations should aim at confirming and extending these findings and at developing translational molecular imaging tools for bridging the gap between basic molecular and cellular neuroscience and clinical research.

Multiparametric magnetic resonance imaging including oxygenation mapping of experimental ischaemic stroke.

Recent advances in MRI methodology, such as microvascular and brain oxygenation (StO2) imaging, may prove useful in obtaining information about the severity of the acute stroke. We assessed the potential of StO2 to detect the ischaemic core in the acute phase compared to apparent diffusion coefficient and to predict the final necrosis. Sprague-Dawley rats (n = 38) were imaged during acute stroke (D0) and 21 days after (D21). A multiparametric MRI protocol was performed at 4.7T to characterize brain damage within three region of interest: 'LesionD0' (diffusion), 'Mismatch' representing penumbra (perfusion/diffusion) and 'Hypoxia' (voxels < 40% of StO2 within the region of interest LesionD0). Voxel-based analysis of stroke revealed heterogeneity of the region of interest LesionD0, which included voxels with different degrees of oxygenation decrease. This finding was supported by a dramatic decrease of vascular and perfusion parameters within the region of interest hypoxia. This zone presented the lowest values of almost all parameters analysed, indicating a higher severity. Our study demonstrates the potential of StO2 magnetic resonance imaging to more accurately detect the ischaemic core without the inclusion of any reversible ischaemic damage. Our follow-up study indicates that apparent diffusion coefficient imaging overestimated the final necrosis while StO2 imaging did not.

Exercise Does Not Protect against Peripheral and Central Effects of a High Cholesterol Diet Given Ad libitum in Old ApoE-/- Mice.

Aim: Advanced atherosclerosis increases inflammation and stroke risk in the cerebral vasculature. Exercise is known to improve cardio-metabolic profiles when associated with a caloric restriction, but it remains debated whether it is still beneficial without the dietary control. The aim of this study was to determine both the peripheral and central effects of exercise training combined with a cholesterol-rich diet given ad libitum in old ApoE-/- mice. Methods: Forty-five-weeks old obese ApoE-/- mice fed with a high cholesterol diet ad libitum were divided into Exercise-trained (EX; running wheel free access) and Sedentary (SED) groups. Insulin tolerance and brain imaging were performed before and after the twelve-weeks training. Tissue insulin resistance, oxidative stress, and inflammation markers in plasma, aorta, and brain were then assessed. Results: In EX ApoE-/- mice, no beneficial effect of exercise was observed on weight, abdominal fat, metabolic parameters, oxidative stress, or inflammation compared to SED. Despite the regular exercise training in ApoE-/- EX mice (mean of 12.5 km/week during 12 weeks), brain inflammation imaging score was significantly associated with increased blood brain barrier (BBB) leakage evaluated by imaging follow-up (r2 = 0.87; p = 0.049) with a faster evolution compared to SED ApoE-/-mice. Conclusion: We conclude that in a context of high cardio-metabolic risk, exercise does not provide any protective effect in old ApoE-/- animals under high cholesterol diet given ad libitum. Peripheral (insulin sensitivity and oxidative/inflammatory status) but also central features (BBB preservation and protection against inflammation) did not show any benefits of exercise. Indeed, there was a fast induction of irreversible brain damage that was more pronounced in exercise-trained ApoE-/- mice.